Purification and some properties of NAD-glycohydrolase from conidia of Neurospora crassa

NAD-glycohydrolase from conidia of Neurospora crassa was purified by affinity chromatography, using 4-methylnicotinamide adenine dinucleotide as ligand immobilized onto Sepharose through a hydrophilic spacer arm. The pure enzyme is a glycoprotein with an isoelectric point of 5.5 and a molecular weight of 33 000 as determined by sodium dodecylsulphate gel electrophoresis. The specific activity is the highest so far found for NAD-glycohydrolases and in various aspects the enzyme is different from that isolated from mycelia of N. crassa grown in a 'zinc-deficient' medium.     

Genetic and biochemical properties of streptococcal NAD-glycohydrolase inhibitor

The gene encoding streptolysin O (slo), a cytolysin of hemolytic streptococci, is transcribed polycistronically from the promoter of the preceding NAD-glycohydrolase (NADase) gene (nga). Between nga and slo, a putative open reading frame (orf1) is located whose function has been totally unknown. Present investigation demonstrated that the orf1 encodes a protein designated as streptococcal NADase inhibitor (SNI). From its nucleotide sequence, SNI was inferred to comprise 161 amino acid residues and the deduced molecular weight was 18,800. This protein was detectable only within cells. Coexpression of SNI was essential for production of streptococcal NADase, and NADase precursor existed as an inactive complex with SNI, in recombinant Escherichia coli. Monomeric NADase and SNI rapidly formed in vitro a stable heterodimer complex in the ratio 1:1, resulting in complete suppression of the hydrolase activity. Unlike other bacterial NADase inhibitors, SNI was thermostable. This protein, coexpressed and complexed with NADase, may protect the producer cocci from exhaustion of NAD.     

The NAD-glycohydrolase (nga) gene of Streptococcus pyogenes

The gene for NAD-glycohydrolase (nga) of group A streptococci (Streptococcus pyogenes) was identified and shown to be located immediately adjacent to the gene for streptolysin O (slo). The nga gene contains 1341 base pairs and encodes a protein of 447 amino acids, including an N-terminal signal peptide. Results from analysis with the polymerase chain reaction indicated that the nga gene is present in all of the strains tested. Functional extracellular NAD-glycohydrolase, also known as NADase, was detected among a wide variety of clinical isolates and known laboratory strains and shown to be present in 72% of 100 strains examined. In contrast, 92% of strains isolated from patients with invasive streptococcal infections were positive for NADase production.     

NAD-dependent inhibition of the NAD-glycohydrolase activity in A549 cells

NAD glycohydrolases are enzymes that catalyze the hydrolisis of NAD to produce ADP-ribose and nicotinamide. Regulation of these enzymes has not been fully elucidated. We have identified an NAD-glycohydrolase activity associated with the outer surface of the plasma membrane in human lung epithelial cell line A549. This activity is negatively regulated by its substrate -NAD but not by -NAD. Partial restoration of NADase activity after incubation of the cells with arginine or histidine, known ADP-ribose acceptors, suggests that inhibition be regulated by ADP-ribosylation. A549 do not undergo to apoptosis upon NAD treatment indicating that this effect be likely mediated by a cellular component(s) lacking in epithelial cells.     

The superantigenic activity of streptococcal pyrogenic exotoxin B is independent of the protease activity

The nature of the mitogenic activity of pyrogenic streptococcal exotoxin B, also known as streptococcal cysteine protease, has been debated in the literature. Streptococcal exotoxin B has been shown to cleave interleukin-1beta precursor and create biologically active interleukin-1beta, a major cytokine mediating inflammation and shock. This activity could mimic the mitogenicity and cytokine release induced by superantigens in lymphocyte stimulating experiments. In this study, the protease activity of streptococcal exotoxin B was irreversibly inhibited by covalent binding of a tripeptide and the superantigenic properties of streptococcal exotoxin B were found not to be influenced by this inactivation. Native as well as protease-inactivated streptococcal exotoxin B was shown to stimulate T-cell proliferation without a need of metabolically active antigen presenting cells. Furthermore, streptococcal exotoxin B-induced T-cell proliferation was shown to require HLA-DQ since addition of HLA-DQ monoclonal antibodies totally inhibited the mitogenic activity of streptococcal exotoxin B, indicating that streptococcal exotoxin B, as other superantigens, makes direct contact with the T-cell receptor via HLA class II. The aim of this study was to characterize the relationship between the proteolytic and superantigenic properties of streptococcal exotoxin B.     

毛花猕猴桃蛋白酶的提纯和性质

用硫酸铵盐析,磷酸盐除果胶和两次ＤＥＡＥ纤维素柱层析等方法,从毛花猕猴桃（ＡｃｔｉｎｉｄｉａｅｒｉａｎｔｈａＢｅｎｔｈ．）无细胞提取液中提纯蛋白酶,在聚丙烯酰胺凝胶电泳上呈现一条带,纯酶的活力为３２５Ｕ／ｍｇ,比活力提高８９倍,总收率约为３７％。用ＳＤＳ聚丙烯酰胺凝胶电泳测定分子量为２３ｋＤ,用等电聚焦电泳测定等电点为４８。酶的最适ｐＨ为３８,最适温度为４３℃左右。纯酶制剂对其底物牛血红蛋白的Ｋｍ值为５５６×１０－３ｍｍｏｌ。酶的紫外吸收光谱最大值为２７８ｎｍ。二巯基苏糖醇、巯基乙醇、Ｌ半胱氨酸盐酸盐等还原剂对该酶有明显的激活作用,而碘乙酸、对氯汞苯甲酸对其有抑制作用,这表明该酶属于巯基酶类。     

Purification and some properties of streptococcal protein G, a novel IgG-binding reagent

Protein G, a bacterial cell wall protein with affinity for immunoglobulin G (IgG), has been isolated from a human group G streptococcal strain (G148). Bacterial surface proteins were solubilized by enzymatic digestion with papain. Protein G was isolated by sequential use of ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and affinity chromatography on Sepharose 4B-coupled IgG. The presence of protein G in various pools and fractions during the isolation was followed by their ability to inhibit the binding of radio-labeled IgG to G148 bacteria. A highly purified protein G was obtained. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the apparent m.w. was 30,000, and on agarose gel electrophoresis the purified protein gave rise to a single band in the alpha 1-region. Protein G was found to bind all human IgG subclasses and also rabbit, mouse, and goat IgG. On the IgG molecule, the Fc part appears mainly responsible for the interaction with protein G, although a low degree interaction was also recorded for Fab fragments. IgM, IgA, and IgD, however, showed no binding to protein G. This novel IgG-binding reagent promises to be of theoretical and practical interest in immunologic research.     

A novel endogenous inhibitor of the secreted streptococcal NAD-glycohydrolase

The Streptococcus pyogenes NAD-glycohydrolase (SPN) is a toxic enzyme that is introduced into infected host cells by the cytolysin-mediated translocation pathway. However, how S. pyogenes protects itself from the self-toxicity of SPN had been unknown. In this report, we describe immunity factor for SPN (IFS), a novel endogenous inhibitor that is essential for SPN expression. A small protein of 161 amino acids, IFS is localized in the bacterial cytoplasmic compartment. IFS forms a stable complex with SPN at a 1:1 molar ratio and inhibits SPN's NAD-glycohydrolase activity by acting as a competitive inhibitor of its beta-NAD+ substrate. Mutational studies revealed that the gene for IFS is essential for viability in those S. pyogenes strains that express an NAD-glycohydrolase activity. However, numerous strains contain a truncated allele of ifs that is linked to an NAD-glycohydrolase-deficient variant allele of spn. Of practical concern, IFS allowed the normally toxic SPN to be produced in the heterologous host Escherichia coli to facilitate its purification. To our knowledge, IFS is the first molecularly characterized endogenous inhibitor of a bacterial beta-NAD(+)-consuming toxin and may contribute protective functions in the streptococci to afford SPN-mediated pathogenesis.     

Basic streptococcal superantigens (SPEX/SMEZ or SPEC) are responsible for the mitogenic activity of the so-called mitogenic factor (MF)

The mitogenic factor (MF) of group A streptococci has been reported to be a superantigen stimulating human T cells carrying Vbeta2, 4 and 8 and has been designated streptococcal pyrogenic exotoxin F (SPEF). MF was also shown to possess DNase activity. Here we have purified MF from culture supernatants of different Streptococcus pyogenes strains. Surprisingly, the MF preparations from different strains showed different Vbeta specificities depending on the expression of SPEC or SMEZ3 by the producing strain. Their mitogenic activity decreased upon further purification. In addition, the mitogenic activity could be only neutralized by antibodies against the basic streptococcal superantigens SPEC or SPEX (SMEZ3) but not by antibodies against MF itself although the latter were able to neutralize completely the DNase activity of MF. We found that streptodornase type B (SDB) was expressed in two molecular forms (SDBI and SDBII), differing only by one additional N-terminal arginine at SDBI. MF was found identical to the enzyme SDBII but is devoid of superantigenic properties and should no longer be called a superantigen or a pyrogenic exotoxin.     

Purification and properties of streptococcal nicotinamide adenine dinucleotide glycohydrolase.

Highly purified streptococcal nicotinamide adenine dinucleotide glycohydrolase (NADase) was obtained by utilizing disodium tetrathionate to protect the enzyme by blocking the sulfhydryl groups of streptococcal proteinase. This was followed by two-step ion-exchange chromatography. The pure enzyme, demonstrated as a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, had a specific activity of 11,200 NADase units per mg of protein and was devoid of hemolytic activity. NADase had a molecular weight of about 55,000 as determined by gel filtration, by summation of amino acid residues, and by sodium dodecyl sulfate/gel electrophoresis. The purified enzyme had optimal activity at pH 7.3 and at 40 C and a calculated Km of 5.1 times 10- minus 4 mM. It was inhibited by alpha-iodoacetamide.     

Purification and properties of a protease inhibitor from crayfish hemolymph

A protease inhibitor from the hemolymph of crayfish, Astacus astacus, has been purified by differential centrifugation, acid precipitation and preparative isoelectric focusing. The inhibitor was apparent homogenous in SDS-electrophoresis and had a molecular weight of 23,000. pI was determined to be 4.7 by isoelectric focusing. No inhibitory activity was lost when the inhibitor was incubated in a pH range of 1–11.5. The purified inhibitor was heat stable. Urea (6 m) had no effect upon the inhibitor. The inhibitor was active against subtilisin and a partly purified protease from the fungus Aphanomyces astaci. Pronase was slightly inhibited whereas trypsin, chymotrypsin, papain, Arthrobacter protease, and extracellular proteases from the fungi Aphanomyces stellatus and A. laevis were unaffected. The importance of protease inhibitors in pathogenesis between the parasitic fungus, A. astaci, and its crayfish host, A. astacus is discussed.     

Macrophage hyperreactivity to endotoxin induced by streptococcal pyrogenic exotoxin in rabbits

Pretreatment of rabbits with streptococcal pyrogenic exotoxin (SPE) resulted in an enhancement of their febrile response to subsequent endotoxin challenge. This suggested that SPE may enhance the macrophage capacity to respond to endotoxin in vivo to produce an endogenous pyrogen. It was also demonstrated that peritoneal macrophages derived from SPE-treated rabbits exhibited hyperreactivity to endotoxin in vitro as assessed by endotoxin-induced increase in glucose consumption. These data indicate that SPE has the ability to enhance macrophage reactivity to endotoxin.     

Purification and some properties of rabbit antiovalbumin

Unlike previous reports that the ovalbumin–anti-ovalbumin complex did not dissociate completely in acid media, our results showed complete dissociation of the complex both in 1.2m-acetic acid, pH2.3, and in KCl–HCl, pH2.2, I 0.06. Thus Sephadex chromatography of the solution obtained by dissolving the antigen–antibody precipitate in these media repeatedly gave two peaks corresponding to anti-ovalbumin and ovalbumin. Further, gel-diffusion and immunoelectrophoresis experiments showed that the phosphate groups of ovalbumin are not involved in the antigenic sites. The antibody thus purified was more easily precipitated than previous preparations. The molecular weight and Stokes radius of the antibody were calculated from its gel-filtration behaviour and were found to be 148000 and 4.8nm respectively. The molecular weight determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis was essentially similar (about 0.7% lower).     

短小芽孢杆菌2080碱性蛋白酶的纯化与性质

短小芽孢杆菌（Bacillus pumilus）2080碱性蛋白酶的发酵液经超滤、硫酸铵沉淀、CM Sepharose Fast Flow和DEAE Sepharose Fast Flow离子交换层析得到了纯化的组分。SDS-PAGE电泳分析显示其分子量约为61kDa。酶学性质研究表明,该纯化酶的最适pH为10．5,最适温度为50℃。     

Purification and properties of a keratinolytic metalloprotease from Microbacterium sp

AIMS: This study was developed to purify and to characterize a keratinolytic protease from the bacterium Microbacterium sp. strain kr10. METHODS AND RESULTS: Enzyme purification was carried out by sequential liquid chromatography on Sephadex G-100 and Q-Sepharose columns. The purification was about 255-fold, with a yield of 34%, as determined with azocasein as substrate. The molecular weight of the enzyme was estimated as 42,000 Da by SDS-PAGE. The enzyme had pH and temperature optima of 7.5 and 50 degrees C respectively. This keratinase was inhibited by EDTA and 1,10-phenanthroline, and analysis of metal content indicates that Zn(2+) and Mg(2+) are present. A 2(2) factorial design was developed to investigate the effect of keratinase and mercaptoacetate concentration on feather keratinolysis. Statistical analysis showed that both variables have a significant effect on hydrolysis of keratin. CONCLUSIONS: A new keratinase produced by Microbacterium sp. was purified and characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: This keratinolytic enzyme offers an interesting potential for the hydrolysis of keratin wastes to be used as feed supplement or bioconversion to added-value products.     

N-氨甲酰水解酶的纯化及性质

从Burkholderiacepecianjut1分离纯化N氨甲酰D氨基酸水解酶(NDase)。实验表明,该酶亚基35KD,最适温度为52℃,最适pH为7.2左右。以N氨甲酰D苯丙氨酸作底物,其米氏常数Km为10.22mmol/L,最大反应速度Vmax为0.27mmol/(L·min)。实验表明二价金属离子对酶活有重要影响。     

Purification and some properties of camel carboxypeptidase B

A carboxypeptidase B-like enzyme was purified 116-fold with a recovery of activity of 29% from a crude extract of camel pancreas by a four-step procedure consisting of two anion exchange chromatographies in succession, gel filtration and hydrophobic interaction chromatography. The enzyme was homogeneous on SDS and non-denaturing gel electrophoresis and on gel isoelectric focusing. Its molecular mass was found to be 31.5 kDa and its isoelectric point was estimated as 6.1. It was active towards a number of substrates that are cleaved by carboxypeptidases B from other species and was also susceptible to inhibition by inhibitors of such enzymes. The camel enzyme showed a pH optimum of 8.0 and it was seen to be a relatively potent kinase in vitro. The enzyme purified in this study was very similar to carboxypeptidases B isolated from other species in size, charge, substrate specificity and susceptibility to inhibition and thus it can be identified as camel carboxypeptidase B.